Genome-wide high throughput analysis of DNA methylation in eukaryotes.
Cytosine methylation is the quintessential epigenetic mark. Two well-established methods, bisulfite sequencing and methyl-DNA immunoprecipitation (MeDIP) lend themselves to the genome-wide analysis of DNA methylation by high throughput sequencing. Here we provide an overview and brief review of these methods. We summarize our experience with MeDIP followed by high throughput Illumina/Solexa sequencing, exemplified by the analysis of the methylated fraction of the Neurospora crassa genome ("methylome"). We provide detailed methods for DNA isolation, processing and the generation of in vitro libraries for Illumina/Solexa sequencing. We discuss potential problems in the generation of sequencing libraries. Finally, we provide an overview of software that is appropriate for the analysis of high throughput sequencing data generated by Illumina/Solexa-type sequencing by synthesis, with a special emphasis on approaches and applications that can generate more accurate depictions of sequence reads that fall in repeated regions of a chosen reference genome.
Pomraning
Kyle R
KR
Center for Genome Research and Biocomputing and Department of Biochemistry and Biophysics, Oregon State University, 2011 ALS Building, Corvallis, OR 97331-7305, USA.
Smith
Kristina M
KM
Freitag
Michael
M
D044503:5-Methylcytosine / Q000032:analysis; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019175:DNA Methylation; D001330:Electronic Data Processing; D005057:Eukaryotic Cells / Q000378:metabolism*; D015723:Gene Library; D047468:Immunoprecipitation; D009492:Neurospora crassa / Q000235:genetics; D015203:Reproducibility of Results; D016415:Sequence Alignment; D012984:Software; D013447:Sulfites / Q000737:chemistry
D013447:Sulfites; D044503:5-Methylcytosine; D004247:DNA; C042345:hydrogen sulfite